The present invention relates to apparatus and methods for the separation of molecules, particularly micromolecules having a molecular mass of less than about 5000 Dalton.
There are increasing numbers of micromolecules being used as food and diet supplements, pharmaceuticals and neutraceuticals. Increasing numbers of vitamins, co-factors, plant and microbial extracts are also being developed and used for human and animal consumption. As many of these compounds are micromolecules (having a molecular mass of less than about 5000 Dalton (Da)), there is a need to develop methods to separate or purify these compounds in a fast and economical manner. Traditional separation methods for micromolecules can alter or denature these compounds. Separation methods for larger molecules typically are not considered suitable for use in micromolecule separation. Furthermore, traditional methods can be quite time consuming, expensive and difficult to scale up commercially.
In the past, a preparative electrophoresis technology for macromolecule separation which utilises tangential flow across a polyacrylamide membrane when a charge is applied across the membrane was used to separate micromolecules. The general design of the earlier system facilitated the purification of proteins and other macromolecules under near native conditions. The technology is bundled into a cartridge comprising several membranes housed in a system of specially engineered grids and gaskets which allow separation of macromolecules by charge and/or molecular weight. The system can also concentrate and desalt/dialyse at the same time. The multi-modal nature of the system allows this technology to be used in a number of other areas especially in the production of biological components for medical use. The technology isolates macromolecules using the duality of charge and size. However, the technology could not be extended to the isolation of molecules below about 5000 Da. This meant that while molecules smaller than 5000 Da could be removed using at least charge-based separation, the resulting target molecule could not be captured.
The separation of micromolecules, molecules deemed to be less than about 5 kDa, was previously thought not to be possible using electrophoresis technology devised to separate macromolecules. This was due to the limit in pore size of membranes normally used in the systems. For example, the smallest cut-off produced in polyaccrylamide membranes is about 5 kDa which will retain any molecule larger than 5 kDa.
There were several problems encountered in the separation of micromolecules using an unmodified electrophoresis system. Difficulty retaining micromolecules in the system has been overcome with the addition of combinations of membranes. However, these membranes themselves posed problems in that they are not designed to retain liquids and can produce large levels of electro-endo-osmosis. The liquid retention problem has been solved by backing the membranes with the hydrogel polyacrylamide membranes, which also helped to reduce the electro-endo-osmosis levels.
It is desirable to have a preparative electrophoresis system which can efficiently and effectively remove micromolecules.
The subject invention overcomes the above limitations and others, and teaches an electrophoresis system, which can be scaled up for preparative applications, which apparatus can efficiently and effectively separate micromolecules.
In accordance with the present invention, there is provided an electrophoresis system which efficiently and effectively separate micromolecules.
Further, in accordance with the present invention, there is provided an apparatus for separating micromolecules by electrophoretic separation, the apparatus comprising:
(a) an anode;
(b) a cathode disposed relative to the anode so as to be adapted to generate an electric field in an electric field area therebetween upon application of a voltage potential between the anode and the cathode;
(c) a separation membrane disposed in the electric field area;
(d) a first restriction membrane disposed between the anode and the separation membrane so as to define a first interstitial volume therebetween;
(e) a second restriction membrane disposed between the cathode and the separation membrane so as to define a second interstitial volume therebetween; and
(f) means adapted to provide a sample constituent in a selected one of the first and second interstitial volumes;
wherein upon application of the voltage potential, a selected separation product is removed from the sample constituent, thorough the separation membrane, and provided to the other of the first and second interstitial volume and wherein a micromolecule is capable of being retained in at least one of the interstitial volumes.
Still further, in accordance with the present invention, there is provided an apparatus for separating micromolecules by electrophoresis, the apparatus comprising:
(a) an anode buffer compartment and a cathode buffer compartment;
(b) electrodes positioned in the buffer compartments;
(c) a first chamber and a second chamber positioned on either side of an ion-permeable separation membrane having a defined molecular mass cut-off, the first chamber and the second chamber being positioned between the anode and the cathode buffer compartments and separated by an ion-permeable restriction membrane positioned on each side of the separation membrane, the restriction membrane(s) allowing flow of ions into and out of the compartments and chambers under the influence of an electric field but substantially restrict movement of at least one micromolecule type from the second chamber into the buffer compartment.
Preferably, the buffer compartments, the first chamber and the second chamber are configured to allow flow of the respective buffer, first and second solutions forming streams. In this form, large volumes can be processed quickly and efficiently. The solutions are typically moved or recirculated through the compartments and chambers from respective reservoirs by pumping means. Peristaltic pumps have been found to be particularly suitable for moving the fluids.
Preferably, the ion-permeable separation membrane has a molecular mass cut-off greater than the molecular mass of the micromolecule to be separated.
An advantage of the present invention is that micromolecules can be separated efficiently and effectively using preparative electrophoresis under near native conditions which results in higher yields and excellent recovery.
Another advantage of the present invention is that the system is suitably used in a number of other areas, especially in the production of biological components for medical use.
Another advantage of the present invention is that the system can be suitably configured to remove biological contaminants at the point of separation.
These and other advantages and benefits of the invention will be apparent to those skilled in the art upon reading and understanding of the following detailed description.